Reid Gilmore

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Papers by Reid Gilmore

A cytosolic reductase pathway is required for efficient N-glycosylation of an STT3B-dependent acceptor site

Journal of Cell Science, 2021

N-linked glycosylation of proteins entering the secretory pathway is an essential modification re... more N-linked glycosylation of proteins entering the secretory pathway is an essential modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here, we used an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could be mimicked by the addition of a membrane-impermeable reducing agent. We identified a hypoglycosylated acceptor site that is adjacent to a cysteine involved in a short-range disulfide. We show that efficient glycosylation at this site is influenced by the cytosolic reductive pathway acting on both STT3A- and STT3B-dependent glycosylation. Our results provide fur...

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Cryo-EM structure of the mammalian Sec61 complex bound to the actively translating wheat germ 80S ribosome

Cryo-EM structure of idle yeast Ssh1 complex bound to the yeast 80S ribosome

In vivo kinetics of membrane protein integration into the yeast endoplasmic reticulum

The FASEB Journal, 2011

Deuterium nuclear magnetic resonance investigation of the effects of proteins and polypeptides on hydrocarbon chain order in model membrane systems

Proceedings of the National Academy of Sciences of the United States of America, Oct 1, 1978

Deuterium nuclear magnetic resonance investigation of the effects of proteins and polypeptides on... more

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Targeting STT3A‐oligosaccharyltransferase with NGI‐1 causes herpes simplex virus 1 dysfunction

The FASEB Journal, Feb 27, 2019

Herpes simplex virus 1 (HSV-1) is a contagious neurotropic herpesvirus responsible for oral lesio... more Herpes simplex virus 1 (HSV-1) is a contagious neurotropic herpesvirus responsible for oral lesions and herpesviral encephalitis. The HSV-1 envelope contains N-glycosylated proteins involved in infection and that are candidate drug targets. NGI-1 is a small-molecule inhibitor of oligosaccharyltransferase (OST) complexes STT3A-OST and STT3B-OST, which catalyze cotranslational and post-translational N-glycosylation, respectively. Because host OSTs attach HSV-1 glycans, NGI-1 might have anti-HSV-1 activity. We evaluated HSV-1 function using NGI-1 and human embryonic kidney 293 knockout lines for OST isoform-specific catalytic and accessory subunits. N-glycosylation of 2 representative envelope proteins (gC and gD) was primarily dependent upon STT3A-OST, but to a large extent replaceable by STT3B-OST. Knockouts impairing STT3A-or STT3B-OST activity, by themselves, did not appreciably affect HSV-1 function (plaque-forming units, normalized to viral particles measured by unglycosylated capsid protein VP5 content). However, with cells lacking STT3B-OST activity (missing the catalytic subunit STT3B or the oxidoreductase subunits magnesium transporter 1/tumor suppressor candidate 3) and thus solely dependent upon STT3A-OST for N-glycosylation, NGI-1 treatment resulted in HSV-1 having cell typedependent dysfunction (affecting infectivity with Vero cells much more than with the 293 lines). Ablation of posttranslational N-glycosylation can therefore make HSV-1 infectivity, and possibly masking of immunogenic peptide epitopes by glycans, highly sensitive to pharmacological inhibition of cotranslational N-glycosylation.-Lu, H.,

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Protein translocation across the endoplasmic reticulum: A tunnel with toll booths at entry and exit

Cell, Nov 19, 1993

ABSTRACT

Intracellular Protein Topogenesis

Springer eBooks, 1986

Editing N-Glycan Site Occupancy with Small-Molecule Oligosaccharyltransferase Inhibitors

Cell chemical biology, Oct 1, 2018

Highlights d NGI-1 is a new OST inhibitor targeting catalytic subunits STT3A and STT3B d NGI-1 an... more Highlights d NGI-1 is a new OST inhibitor targeting catalytic subunits STT3A and STT3B d NGI-1 analogs were tested with reporters in STT3A-and STT3B-deficient cells d Analysis revealed inhibitors with differential subunit profiles toward STT3A and STT3B d A series of STT3B-specific inhibitors were identified

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Mutations in <i>MAGT1</i> lead to a glycosylation disorder with a variable phenotype

Proceedings of the National Academy of Sciences of the United States of America, Apr 29, 2019

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Signal recognition particle receptor-β (SR-β) coordinates cotranslational N-glycosylation

Science Advances, Mar 17, 2023

Proteins destined for the secretory compartment of the cell are cotranslationally translocated in... more Proteins destined for the secretory compartment of the cell are cotranslationally translocated into the endoplasmic reticulum. The majority of these proteins are N-glycosylated, a co- and posttranslational modification that ensures proper protein folding, stability, solubility, and cellular localization. Here, we show that the β subunit of the signal recognition particle receptor (SR) is required for assembly of the N-glycosylation–competent translocon. We report that guanine analog chemical probes identified by high-throughput screening or mutation of the SR- β guanosine triphosphate binding site cause an N-glycosylation–deficient phenotype. Neither method alters the association of SR- α with SR- β , but both approaches reduce the association of SR- β with the oligosaccharyltransferase complex. These experiments demonstrate that SR- β has a previously unrecognized function coordinating endoplasmic reticulum translation with N-glycosylation.

Protein translocation across the endoplasmic reticulum. II. Isolation and characterization of the signal recognition particle receptor

Journal of Cell Biology, Nov 1, 1982

Salt-extracted microsomal membranes (K-RM) contain an activity that is capable of releasing the s... more

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The function of signal recognition particle and signal recognition particle receptor in the process of protein translocation across the endoplasmic reticulum membrane

Identification of an integral membrane protein that can be cross linked to nascent polypeptides undergoing translocation across the endoplasmic reticulum

The synthesis of novel N‐glycans in Entamoeba histolytica and the quality control, secretion and cell surface arrangement of glycoproteins

The FASEB Journal, Mar 1, 2006

Entamoeba histolytica is a protozoan parasite, which causes ulcerative colitis and liver disease ... more

Lack of NKG2D in MAGT1-deficient patients is caused by hypoglycosylation

Human Genetics, Feb 19, 2022

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Repository Citation

Dual recognition of the ribosome and the signal recognition particle by the SRP receptor during p... more

JCB: Article

An interaction between the SRP receptor and the translocon is critical during cotranslational pro... more

REVIEW An evolving view of the eukaryotic oligosaccharyltransferase

An evolving view of the eukaryotic oligosaccharyltransferase

Rapid inactivation of the yeast Sec complex selectively blocks transport of post-translationally translocated proteins

Journal of Biological Chemistry, 2021

The yeast endoplasmic reticulum has three distinct protein translocation channels. The heterotrim... more The yeast endoplasmic reticulum has three distinct protein translocation channels. The heterotrimeric Sec61 and Ssh1 complexes, which bind translating ribosomes, mediate cotranslational translocation of proteins targeted to the endoplasmic reticulum by the signal recognition particle (SRP) and SRP receptor targeting pathway, whereas the heptameric Sec complex has been proposed to mediate ribosome-independent post-translational translocation of proteins with less hydrophobic signal sequences that escape recognition by the SRP. However, multiple reports have proposed that the Sec complex may function cotranslationally and be involved in translocation or integration of SRP-dependent protein translocation substrates. To provide insight into these conflicting views, we induced expression of the tobacco etch virus protease to achieve rapid inactivation of the Sec complex by protease-mediated cleavage within the cytoplasmic domain of the Sec63 protein. Protein translocation assays conducted after tobacco etch virus protease induction revealed a complete block in translocation of two well-characterized substrates of the Sec complex, carboxypeptidase Y (CPY) and Gas1p, when the protease cleavage sites were located at structural domain boundaries in Sec63. However, integration of SRP-dependent membrane protein substrates was not detectably impacted. Moreover, redirecting CPY to the cotranslational pathway by increasing the hydrophobicity of the signal sequence rendered translocation of CPY insensitive to inactivation of the Sec complex. We conclude that the Sec complex is primarily responsible for the translocation of yeast secretome proteins with marginally hydrophobic signal sequences.

Reid Gilmore

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